CN103276048A - Preparation method of sample for conveniently and rapidly detecting deoxyribonucleic acid (DNA) cell damage and kit using same - Google Patents
Preparation method of sample for conveniently and rapidly detecting deoxyribonucleic acid (DNA) cell damage and kit using same Download PDFInfo
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Abstract
The invention discloses a preparation method of a sample for conveniently and rapidly detecting deoxyribonucleic acid (DNA) cell damage. The method mainly comprises a gel sheet-making step, a cell lysis step, a DNA melting step, an electrophoresis step, a neutralizing step and a dyeing step, wherein in the gel sheet-making step, two layers of gels need to be spread; and in the dyeing step, a GelRed nucleic acid gel dye with high sensitivity and low toxicity is used. The invention also provides a kit using the method. The kit comprises normal melting-point agarose, low melting-point agarose, a cell lysis solution, an electrophoretic buffer solution, the DNA gel dye and a frosted edgeglass slide. According to the method, fluorescence dyeing is carried out by using the GelRed nucleic acid gel dye, and the GelRed nucleic acid gel dye is high in sensitivity, low in toxicity and stable, and environment pollution cannot be caused by wastes, so that the method is safe and environment-friendly; and because of only two layers of the spread gels, compared with a sandwich gel-spreading method used in the traditional comet assay, the preparation method is easy to operate and difficult to degum, and is uniform in dyeing; and moreover, the obtained electrophoresis image is relatively clear and objective.
Description
Technical field
The present invention relates to DNA in mammalian cells damage check technology, particularly a kind of for the preparation method of comet experiment test sample and the test kit that is applied to this method.
Background technology
DNA is storing the genetic information that organism depends on for existence and multiplies.The cell DNA damage is one of focus of cytobiology area research.When cell sustained damage, its dna double chain can rupture, and specificity cascade biochemical reaction can take place in the process, relied on Ca
2+, Mg
2+Endonuclease be activated, act preferentially on zone between the nucleosome that connects DNA, the DNA chain is cut into the fragment of 180～200bp or its multiple, DNA travelling speed and migration distance that fracture takes place in the comet electrophoresis are several times as much as Normocellular nuclear DNA, present and break away from nuclear " comet " shape, DNA damaged cell and normal cell can be distinguished accordingly.
Comet electrophoresis detection method (Comet Assay), initiated in 1984 by Ostling, it is a kind of method that detects the eukaryotic cell dna damage in unicellular level, the mensuration of the indexs such as length of the dna content per-cent of comet occurrence rate, the tail of a comet, the tail of a comet by to cell electrophoresis the time can cause that the cell DNA damage makes comprehensive evaluation to cell drug.1993, Olive etc. were applied to Using Comet Assay apoptotic detection first.Based on principle be, eukaryotic dna molecular amount is bigger, and DNA is negative superhelix structure and is attached in the nuclear matrix, adopt sepharose with cell embedding under cell pyrolysis liquid, cytolemma, nuclear membrane and other biological film destroy make intracellular RNA, protein and other compositions enter gel.Then be diffused in the lysate, and nuclear DNA still keeps twining shape, is attached on the remaining nuclear skeleton, and stays original position.When highly basic (pH=13) electrophoresis liquid electrophoresis, if cell does not sustain damage, nuclear dna molecular amount still rests in the nuclear matrix greatly and because fracture does not take place, and behind fluorescent dyeing, nuclear forms a bright head, no conditions of streaking in position; If dna single chain or double-strand break appear in the impaired meeting of cell, DNA untwists and is strand under the strong alkali environment, the dna break fragment can enter in the gel, under the electrical forces effect, the dna fragmentation of these fractures moves because carrying negative charge disengaging nuclear DNA anode, the DNA fragment forms the comet formation afterbody, can form the comet image.
Comet electrophoresis detection method is usually used in detecting the degree of impairment of cell DNA, also can judge the dead mode of cell according to the form of comet.This detection method has its unique advantage: required sample size is few, be applicable to any eukaryotic cell, highly sensitive, the cell of research need not be in m period, need not advantages such as radio-labeling, be widely used in the eukaryotic cell dna damage and repaired the research of environmental organism monitoring, drug screening, tumor invasion mechanism and treatment and Apoptosis Mechanism.At present, the specimen preparation process that is used for the analysis of comet electrophoresis detection mainly comprises: sample preparing gel, lysis, untwist and electrophoresis, neutralization washing, fluorescent dye and adopt comet gel electrophoresis camera system to take fluorescence photo.Usually, in the fluorescent dye step employed fluorescence dye be ethidium bromide (Ethidium Bromide, EB), and EB is a kind of strong mutagenic compound, evaporates in the air easily, and test operation personnel's health is threatened; Dyeing slide after the use is directly thrown into rubbish container may cause potential hazard to surrounding environment.In addition, the existing gel slide that is used for the comet electrophoretic analysis need the 3 layers of gel in shop, and in follow-up cracking, electrophoresis and rinse cycle, gel very easily breaks away from slide glass, causes the glue failure, has influenced the promotion and application of comet electrophoretic technique.
Summary of the invention
Purpose of the present invention, be the defective that exists in the existing detection technique is improved, a kind of easy, quick, highly sensitive, nontoxic method that is used for the specimen preparation of mammalian cell detected by comet assay is provided, and provides a kind of for the test kit of implementing aforesaid method.
The technical solution adopted in the present invention is: a kind of method of easy, rapid detection cell DNA damage specimen preparation, its preparation process comprise mainly that gel film-making, lysis, DNA unwind, electrophoresis, neutralization dehydration and fluorescent dye.Wherein said gel film-making step need be spread 2 layers of gel, and described staining procedure adopts highly sensitive, hypotoxic Gel Red nucleic acid gel dyestuff.
Described gel film-making step, be with 100 μ L, 1% normal fusing point agarose drop to the slide glass of frosted limit, evenly push open with another piece slide, place 4 ℃ of refrigerators to solidify immediately after the covered rapidly 10 minutes, obtain the first layer gel; 60 μ L cell suspension to be measured is mixed by the 1:2 volume ratio in 42 ℃ of water-baths with 0.78% low normal fusing point agarose, drip the above-mentioned mixed solution of 80 μ L to the first layer glue, covered places 4 ℃ of refrigerators to solidify 10 minutes, obtains being covered with the slide of two-layer gel.Described frosted limit slide glass is the slide that a side has even frosting.Described 1% normal fusing point sepharose (NMA) and 0.78% low normal fusing point sepharose (LNA) all use 0.01M phosphoric acid buffer (PBS) formulated.
Described lysis step is after walking cover glass with the careful translation of elbow tweezers, the slide that is covered with two-layer gel is immersed be equipped with in the plate of precooling cell pyrolysis liquid, 4 ℃ of lucifuge cracking 60 minutes.Take out slide glass, with distilled water rinsing 2 times gently, obtain the slide after the lysis.Described cell pyrolysis liquid be by the Tris damping fluid (10mM, pH=10), massfraction is that 1% sodium sarcosinate, volume fraction are that 1% Triton X-100, volume fraction are that 10% dimethyl sulfoxide (DMSO) (DMSO) is formed; Described Tris damping fluid is by 10mM Trizma base, 100mMNa
2EDTA, 2.5M NaCl form; Described Triton X-100 and DMSO use preceding adding facing.
Described DNA unwinds and electrophoresis step, is the slide after the above-mentioned lysis is placed horizontal strip electrophoresis groove positive terminal, slowly pours the electrophoretic buffer of new preparation into, covers approximately about slide glue face 0.25cm, and lucifuge left standstill after 30 minutes carries out electrophoresis.Described electrophoretic buffer is by 1mMNa
2EDTA and 300mM NaOH are formulated.Described electrophoretic voltage is 26v, and electric current is 300mA, and electrophoresis time is 20 minutes.
In described and dehydrating step, be that the slide behind the electrophoresis is immersed Tris-HCl neutralization buffer (0.4mM, pH=7.5), embathe 3 times, use 0.01M PBS to clean, slide is the dehydration of 50%, 75%, 100% ethanol gradient through volume fraction successively, each 2 minutes, dry the slide after obtaining neutralizing under the room temperature.
Described fluorescent dye step is Dropwise 50 μ L Gel Red fluorescence dye on the slide after the above-mentioned dehydration, pushes Gel Red gently open with cover glass, and Gel Red is evenly spread out on the agarose surface, covered, lucifuge dyeing 10 minutes.
The present invention is provided with quick detection kit, comprises slide glass and the following reagent for preparing that 6 sides have even frosting:
(1) 1% normal fusing point agarose
(2) 0.78% low normal fusing point agaroses
(3) cell pyrolysis liquid
(4) electrophoretic buffer
(5) neutralization buffer
(6) fluorescence dye
(7) gradient alcohol.
Wherein, being formulated as follows of described reagent:
(1) 1% normal fusing point agarose: formulated with 0.01M phosphoric acid buffer (PBS);
(2) 0.78% low normal fusing point agaroses: formulated with 0.01M PBS;
(3) cell pyrolysis liquid: the dimethyl sulfoxide (DMSO) (DMSO) by 10mM Tris damping fluid (pH=10), massfraction 1% sodium sarcosinate, volume fraction 1%Triton X-100, volume fraction 10% is formed; Described Tris damping fluid is by 10mM Trizma base, 100mMNa
2EDTA, 2.5M NaCl form, and wherein said Triton X-100 and DMSO use preceding adding facing;
(4) electrophoretic buffer: by 1mM Na
2EDTA and 300mM NaOH form;
(5) neutralization buffer: be 0.4mM Tris-HCl neutralization buffer, pH=7.5;
(6) fluorescence dye: be Gel Red nucleotide fluorescent dye;
(7) gradient alcohol: be that 50%, 75%, 100% ethanol is formed by volume fraction.
The present invention compared with prior art has following advantage and effect:
(1) gel film-making step involved in the present invention only needs the 2 layers of glue in shop, will spread " sandwich " shop adhesive process of 3 layers of glue with traditional comet laboratory sample slide and compare, and is difficult for coming unstuck, and is workable, is easy to promote.
(2) the used slide glass of the present invention is the slide that a side has even frosting, efficiently solves the degumming problem that often runs in the glue process of shop, can obtain more firm gel, has improved shop glue success ratio.
(3) the present invention adopts Gel Red nucleic acid gel dyestuff to carry out fluorescent dye, because its low toxicity, stable, (AO, EB, PI) compares with the conventional fluorescent dyestuff, and be little to the toxicity of mammalian cell, and waste can not cause environmental pollution.
(4) the present invention adopts gradient alcohol that sample is dewatered, and can make the sample dehydration more thorough, when carrying out the analysis of comet electrophoretic image, has weakened the fluorescence decay degree, has improved picture quality.
(5) the invention provides test kit for the DNA in mammalian cells damage check, can detect the DNA in mammalian cells degree of injury easily and quickly, whole testing process can be finished within 3 hours.
Description of drawings
Fig. 1 is to adopt method of the present invention at different concns ionic liquid [C
8Mim] the Br effect down, the Using Comet Assay electrophoretic image of HepG2 cell sample slide.
Embodiment
Below the present invention will be further described by implementing example, but embodiments of the present invention are not limited thereto.
(1) human liver cancer cell Heptatoma G2(HepG2) cultivates in the DMEM nutrient solution of 10% foetal calf serum, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, routinely 37 ℃ of attached cells, 5%CO
2Cultivate in the incubator.Discard nutrient solution during passage, PBS washes 2 times, 0.25% trysinization of HepG2 cell, and regulating cell concn is 1 * 10
6Individual/ml is inoculated in and cultivates 24 hours in 6 orifice plates to 80% degrees of fusion, different concns 1-butyl-3-Methylimidazole bromine ([C
8Mim] Br) ionic liquid effect 24 hours, other establishes the blank group.After drug effect finishes, PBS washing 2 times, centrifugal 5 minutes collecting cells of 800r/min are resuspended in 1ml precooling PBS with cell, measure cell survival rate with the trypan blue method〉95%, adjusting cell density is (1～5) * 10
5/ mL is standby.
(2) the first layer Jiao Pu system.The normal fusing point agarose drop of 100 μ L1% to the slide glass of frosted limit, is evenly pushed open with another piece slide, placed 4 ℃ of refrigerators to solidify immediately after the covered rapidly 10 minutes, obtain the first layer gel.
(3) second layer Jiao Pu system.60 μ L cell suspension to be measured is mixed by the 1:2 volume ratio in 42 ℃ of water-baths with 0.78% low normal fusing point agarose, drip the above-mentioned mixed solution of 80 μ L to the first layer glue, covered places 4 ℃ of refrigerators to solidify 10 minutes.Obtain being covered with the slide of two-layer gel.
(4) lysis.After walking cover glass with the careful translation of elbow tweezers, the slide that is covered with two-layer gel immersed be equipped with in the plate of precooling cell pyrolysis liquid, 4 ℃ of lucifuge cracking 60 minutes.Take out slide glass, with distilled water rinsing 2 times gently, obtain the slide after the lysis.Described cell pyrolysis liquid be by the Tris damping fluid (10mM, pH=10), massfraction is that 1% sodium sarcosinate, volume fraction are that 1% Triton X-100, volume fraction are that 10% dimethyl sulfoxide (DMSO) (DMSO) is formed; Described Tris damping fluid is by 10mM Trizma base, 100mMNa
2EDTA, 2.5M NaCl form; Described Triton X-100 and DMSO use preceding adding facing.
(5) DNA unwinds and electrophoresis.Slide after the above-mentioned lysis is placed horizontal strip electrophoresis groove positive terminal, slowly pour the electrophoretic buffer of new preparation into, covered approximately about slide glue face 0.3cm, lucifuge left standstill 30 minutes, was 25V at electrophoretic voltage, and electric current is 300mA, carries out electrophoresis 20 minutes.Obtain the slide behind the electrophoresis.Described electrophoretic buffer is by 1mM Na
2EDTA and 300mM NaOH are formulated.
(6) neutralization dehydration.Slide behind the electrophoresis is immersed the Tris-HCl neutralization buffer, and (0.4mM pH=7.5), embathes 3 times, use 0.01M PBS to clean, slide is the dehydration of 50%, 75%, 100% ethanol gradient through volume fraction successively, each 2 minutes, dry the slide after obtaining neutralizing under the room temperature.
(7) fluorescent dye.Dropwise 50 μ L Gel Red fluorescence dye is pushed Gel Red open gently with cover glass on the slide after the above-mentioned dehydration, and Gel Red is evenly spread out on the agarose surface, and covered, lucifuge dyeed about 10 minutes.
(8) microscopy is taken pictures.5 sample slides making and 1 blank slide placed 515～560nm is excitation wavelength under the Lycra fluorescent microscope, observe with " 100 * " object lens, each sample is selected 50～100 cells pickup image of taking pictures at random.
(9) result's statistics and analysis.50 hangovers of each sample slide picked at random cell adopts CASP1.2.2 comet analysis software to measure comet tail long (Tail length), comet tail force square (Tail moment), comet tail DNA percentage composition (Tail DNA%).Experimental data adopts Origin7.5 software to carry out ANNOVA and analyzes, and drug effect group and control group are carried out significant t-test, with p＜0.05 as the significance foundation, thereby obtain HepG2 cell sample apoptosis and DNA chain chain rupture degree of injury.
As shown in Figure 1, adopt the mammalian cell Using Comet Assay sample of present method preparation, can obtain clear, objective electrophoretic image, and have very high fluorescent brightness.Image result shows that the cell DNA electrophoresis of damage shows as the increase of tail of a comet length and afterbody fluorescence strengthens, and forms typical comet image.Measure comet tail long (Tail length), comet tail force square (Tail moment), comet tail DNA percentage composition (TailDNA%) by adopting CASP 1.2.2 comet analysis software.Adopt SPSS software to analyze as shown in table 1 to HepG2 cell sample DNA chain chain rupture degree of injury:
Table 1. different concns [C
8Mim] following HepG2 cell DNA damage dose---the effect relation of Br effect
Annotate:
﹡ ﹡P ﹤ 0.01(and control group 0mmol/L[C
8Mim] Br compares)
The result shows that along with the increase of drug level, HepG2 cell DNA degree of injury also increases, and shows significant concentration---effect relation.
Claims (8)
1. the sample preparation methods of easy, a rapid detection cell DNA damage is characterized in that comprising that gel film-making, lysis, DNA unwind, the step of electrophoresis, neutralization dehydration and fluorescent dye, in the wherein said gel film-making step, only needs to spread 2 layers of glue; Used slide glass is the slide that a side has even frosting; Described fluorescent dye process adopts Gel Red nucleic acid gel dyestuff; In the described dehydrating step, adopt gradient alcohol that sample is dewatered.
2. the sample preparation methods for detection of cell DNA damage according to claim 1, it is characterized in that, described gel film-making step, be with 100 μ L, 1% normal fusing point agarose drop to the slide glass of frosted limit, evenly push open with another piece slide, place 4 ℃ of refrigerators to solidify immediately after the covered rapidly 10 minutes, obtain the first layer gel; 60 μ L cell suspension to be measured is mixed by the 1:2 volume ratio in 42 ℃ of water-baths with 0.78% low normal fusing point agarose again, drip the above-mentioned mixed solution of 80 μ L to the first layer glue, covered places 4 ℃ of refrigerators to solidify 10 minutes, obtains being covered with the slide of two-layer gel.
3. method according to claim 1 is characterized in that, described lysis process is to walk cover glass with the careful translation of elbow tweezers, the slide that is covered with two-layer gel is immersed be equipped with in the plate of precooling cell pyrolysis liquid, 4 ℃ of lucifuge cracking 60 minutes; Take out slide glass, with distilled water rinsing 2 times gently, obtain the slide after the lysis.
4. method according to claim 1, it is characterized in that, described DNA unwinds, electrophoresis process, be that the slide after the above-mentioned lysis is placed horizontal strip electrophoresis groove positive terminal, slowly pour the electrophoretic buffer of new preparation into, covered approximately about slide glue face 0.3cm, lucifuge left standstill 30 minutes, be 25V at electrophoretic voltage, electric current is 300mA, carries out electrophoresis 20 minutes.
5. the sample preparation methods for detection of cell DNA damage according to claim 1, it is characterized in that, in described and dehydration, be the Tris-HCl neutralization buffer that the slide behind the electrophoresis is immersed 0.4mM, pH=7.5, embathe 3 times, use 0.01M PBS to clean, slide is the dehydration of 50%, 75%, 100% ethanol gradient through volume fraction successively, each 2 minutes, dry the slide after obtaining neutralizing under the room temperature.
6. the sample preparation methods for detection of cell DNA damage according to claim 1, it is characterized in that, described fluorescent dye process, it is Dropwise 50 μ L Gel Red fluorescence dye on the slide after the above-mentioned dehydration, push Gel Red gently open with cover glass, Gel Red is evenly spread out on the agarose surface, and covered, lucifuge dyeed about 10 minutes.
7. detection kit that is used for the described method of claim 1 is characterized in that it comprises slide glass and the following reagent for preparing that 6 sides have even frosting:
(1) 1% normal fusing point agarose
(2) 0.78% low normal fusing point agaroses
(3) cell pyrolysis liquid
(4) electrophoretic buffer
(5) neutralization buffer
(6) fluorescence dye
(7) gradient alcohol.
8. test kit according to claim 7 is characterized in that, being formulated as follows of described reagent:
(1) 1% normal fusing point agarose: formulated with the 0.01M phosphoric acid buffer;
(2) 0.78% low normal fusing point agaroses: formulated with 0.01M PBS;
(3) cell pyrolysis liquid: the dimethyl sulfoxide (DMSO) by the 10mM Tris damping fluid of pH=10, massfraction 1% sodium sarcosinate, volume fraction 1% Triton X-100, volume fraction 10% is formed; Described Tris damping fluid is by 10mM Trizma base, 100mM Na
2EDTA, 2.5M NaCl form, and wherein said Triton X-100 and dimethyl sulfoxide (DMSO) are used preceding adding facing;
(4) electrophoretic buffer: by 1mM Na
2EDTA and 300mM NaOH form;
(5) neutralization buffer: be 0.4mM Tris-HCl neutralization buffer, pH=7.5;
(6) fluorescence dye: be Gel Red nucleotide fluorescent dye;
(7) gradient alcohol: be that 50%, 75%, 100% ethanol is formed by volume fraction.
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| CN106018529A (en) * | 2016-07-07 | 2016-10-12 | 中国科学院重庆绿色智能技术研究院 | Kit using single cell gel electrophoresis for detecting cell DNA damage and method |
| CN106568824A (en) * | 2016-11-08 | 2017-04-19 | 浙江大学 | Method for detecting DNA damage of mussels under stress of compound heavy metals |
| CN107576713A (en) * | 2017-07-12 | 2018-01-12 | 浙江省农业科学院 | A kind of method for detecting earthworm coelomocyte DNA damage and its utilization |
| CN108896750A (en) * | 2018-05-11 | 2018-11-27 | 江苏大学 | A kind of preparation method and purposes of BSA-Au/Ag NCs/OPD/HRP proportional-type fluorescent optical sensor |
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| CN113029732A (en) * | 2021-03-09 | 2021-06-25 | 中南大学 | Operation device and operation method for single cell gel electrophoresis experiment |
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| CN105044191A (en) * | 2015-07-24 | 2015-11-11 | 哈尔滨工业大学 | Comet assay method for cell gene damage detection of fish muscle tissue |
| CN106018529A (en) * | 2016-07-07 | 2016-10-12 | 中国科学院重庆绿色智能技术研究院 | Kit using single cell gel electrophoresis for detecting cell DNA damage and method |
| CN106568824A (en) * | 2016-11-08 | 2017-04-19 | 浙江大学 | Method for detecting DNA damage of mussels under stress of compound heavy metals |
| CN107576713A (en) * | 2017-07-12 | 2018-01-12 | 浙江省农业科学院 | A kind of method for detecting earthworm coelomocyte DNA damage and its utilization |
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| CN110470520A (en) * | 2019-09-16 | 2019-11-19 | 武汉赛维尔生物科技有限公司 | Liquid method is changed in a kind of comet |
| CN110849847A (en) * | 2019-10-21 | 2020-02-28 | 中国药科大学 | Cell membrane damage quick response sensor and preparation method and application thereof |
| CN110849847B (en) * | 2019-10-21 | 2022-05-10 | 中国药科大学 | Cell membrane damage quick response sensor and preparation method and application thereof |
| CN113029732A (en) * | 2021-03-09 | 2021-06-25 | 中南大学 | Operation device and operation method for single cell gel electrophoresis experiment |
| CN113405881A (en) * | 2021-06-10 | 2021-09-17 | 华东理工大学 | Rapid detection method and kit for single cell DNA damage |
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Application publication date: 20130904 |